A High-Content Screen Identifies Drugs That Restrict Tumor Cell Extravasation across the Endothelial Barrier.

Metastases largely rely on hematogenous dissemination of tumor cells via the vascular system and significantly limit prognosis of patients with solid tumors. To colonize distant sites, circulating tumor cells must destabilize the endothelial barrier and transmigrate across the vessel wall. Here we performed a high-content screen to identify drugs that block tumor cell extravasation by testing 3,520 compounds on a transendothelial invasion coculture assay. Hits were further characterized and validated using a series of in vitro assays, a zebrafish model enabling three-dimensional (3D) visualization of tumor cell extravasation, and mouse models of lung metastasis. The initial screen advanced 38 compounds as potential hits, of which, four compounds enhanced endothelial barrier stability while concurrently suppressing tumor cell motility. Two compounds niclosamide and forskolin significantly reduced tumor cell extravasation in zebrafish, and niclosamide drastically impaired metastasis in mice. Because niclosamide had not previously been linked with effects on barrier function, single-cell RNA sequencing uncovered mechanistic effects of the drug on both tumor and endothelial cells. Importantly, niclosamide affected homotypic and heterotypic signaling critical to intercellular junctions, cell-matrix interactions, and cytoskeletal regulation. Proteomic analysis indicated that niclosamide-treated mice also showed reduced levels of kininogen, the precursor to the permeability mediator bradykinin. Our findings designate niclosamide as an effective drug that restricts tumor cell extravasation through modulation of signaling pathways, chemokines, and tumor-endothelial cell interactions. SIGNIFICANCE: A high-content screen identified niclosamide as an effective drug that restricts tumor cell extravasation by enhancing endothelial barrier stability through modulation of molecular signaling, chemokines, and tumor-endothelial cell interactions. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/3/619/F1.large.jpg.

Changes in the Glycosylation of Kininogen and the Development of a Kininogen-Based Algorithm for the Early Detection of HCC.

Background: Hepatocellular carcinoma (HCC) has the greatest increase in mortality among all solids tumors in the United States related to low rates of early tumor detection. Development of noninvasive biomarkers for the early detection of HCC may reduce HCC-related mortality.Methods: We have developed an algorithm that combines routinely observed clinical values into a single equation that in a study of >3,000 patients from 5 independent sites improved detection of HCC as compared with the currently used biomarker, alpha-fetoprotein (AFP), by 4% to 20%. However, this algorithm had limited benefit in those with AFP <20 ng/mL. To that end, we have developed a secondary algorithm that incorporates a marker, fucosylated kininogen, to improve the detection of HCC, especially in those with AFP <20 ng/mL and early-stage disease.Results: The ability to detect early-stage AFP-negative (AFP <20 ng/mL) HCC increased from 0% (AFP alone) to 89% (for the new algorithm). Glycan analysis revealed that kininogen has several glycan modifications that have been associated with HCC, but often not with specific proteins, including increased levels of core and outer-arm fucosylation and increased branching.Conclusions: An algorithm combining fucosylated kininogen, AFP, and clinical characteristics is highly accurate for early HCC detection.Impact: Our biomarker algorithm could significantly improve early HCC detection and curative treatment eligibility in patients with cirrhosis. Cancer Epidemiol Biomarkers Prev; 26(5); 795-803. ©2017 AACR.

Adsorption of plasma proteins on uncoated PLGA nanoparticles.

The biodistribution of nanoparticles is significantly influenced by their interaction with plasma proteins. In order to optimize and possibly monitor the delivery of drugs bound to nanoparticles across the blood-brain barrier (BBB), the protein adsorption pattern of uncoated poly(lactic-co-glycolic acid) (PLGA) nanoparticles after their incubation in human plasma was studied by mass spectrometry. After washing of the particles with water, the proteins were directly digested on the nanoparticle surface using trypsin and then analyzed by nLC MALDI-TOF/TOF. Up to now, the standard method for investigation into the plasma protein adsorption to the particles was 2D gel electrophoresis (2D-PAGE), in certain cases followed by mass spectrometry. The non-gel-based method proposed in the present study provides novel insights into the protein corona surrounding the nanoparticles. The proteins adsorbed on the PLGA nanoparticles after incubation that gave the best signal in terms of quality (high MASCOT score) in human plasma were apolipoprotein E, vitronectin, histidine-rich glycoprotein and kininogen-1. These proteins also are constituents of HDL.

[Direct proteomic profiling of human urine and blood serum in an experiment with 5-day dry immersion].

Changes in proteome of urine and blood serum obtained from 14 healthy humans (age 21-29 yrs) medically certified for an experiment with dry immersion were analyzed. Urine and serum samples were pre-fractionated and enriched with magnetic particles MB-WCX and MB-HIC, respectively, on robot ClinProt (Bruker Daltonics) for direct mass-spectrometry profiling by MALDI-TOF. As a result, 143 protein peaks on the average were identified in urine samples. It was shown that a high variation coefficient in 23.7% of protein peaks, i.e. double technical, points to the most plastic fraction of the urine proteome. In blood serum, 175 peaks were identified in a sample on the average. Comparison of baseline and immersion mass-spectra of the blood proteome revealed significant differences. Increased peak areas of several protein fragments--C3 and C4 fragments of complement system, high-molecular kininogen and fibrinogen--can be ascribed to human body adaptation to the experimental conditions.

[Direct proteome profiling of human blood serum in the experiment with 5-day dry immersion].

Purpose of the investigation was to determine changes in blood plasma proteome in healthy human subjects (n = 14, 19 to 26 y.o.) in an experiment with dry immersion (DI). Plasma samples were drawn 7 and 2 days before the exposure, on DI days 2, 3 and 5, and on days 1, 3, 7 and 15 after the experiment. Previous to direct MALDI-TOF mass-spectrometric profiling, serum samples were pre-fractionated and enriched with magnetic particles MB WCX (WCX--a weak cation exchanger) on ClinProt (Bruker Daltonics). In each spectrum, 175 MS-peaks were detected on average within the mass range from 1000 to 17,000 Da with the signal/noise ratio = 5. Student's criterion (p < 0.05) was used to define reliable differences between DI and baseline samples from 48 peaks (27.4 % of all the proteome profile peaks). On DI days 2 and 3, growth of peak areas was observed in fragments of complement system proteins C3 and C4, high-molecular kininogen and fibrinogen that can be attributed to organism adaptation to conditions of the experiment. Significant increases of the peak area of apolipoprotein CI (reduced form with segregated threonine and proline) and C4 enzymes of the complement system, and fibrinogen on the first day after the experiment can be related to changes in motor activities of the subjects.

Identification and characterization of novel ERC-55 interacting proteins: evidence for the existence of several ERC-55 splicing variants; including the cytosolic ERC-55-C.

ERC-55, encoded from RCN2, is localized in the ER and belongs to the CREC protein family. ERC-55 is involved in various diseases and abnormal cell behavior, however, the function is not well defined and it has controversially been reported to interact with a cytosolic protein, the vitamin D receptor. We have used a number of proteomic techniques to further our functional understanding of ERC-55. By affinity purification, we observed interaction with a large variety of proteins, including those secreted and localized outside of the secretory pathway, in the cytosol and also in various organelles. We confirm the existence of several ERC-55 splicing variants including ERC-55-C localized in the cytosol in association with the cytoskeleton. Localization was verified by immunoelectron microscopy and sub-cellular fractionation. Interaction of lactoferrin, S100P, calcyclin (S100A6), peroxiredoxin-6, kininogen and lysozyme with ERC-55 was further studied in vitro by SPR experiments. Interaction of S100P requires [Ca(2+)] of approximately 10(-7) M or greater, while calcyclin interaction requires [Ca(2+)] of >10(-5) M. Interaction with peroxiredoxin-6 is independent of Ca(2+). Co-localization of lactoferrin, S100P and calcyclin with ERC-55 in the perinuclear area was analyzed by fluorescence confocal microscopy. The functional variety of the interacting proteins indicates a broad spectrum of ERC-55 activities such as immunity, redox homeostasis, cell cycle regulation and coagulation.

Vitreous proteomic analysis of proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is the most common cause of anatomic failure in retinal detachment surgery. To understand the molecular mechanisms, vitreous proteomes of patients with PVR were investigated by two-dimensional-nano-liquid chromatography coupled with tandem mass spectrometry. Vitreous samples of moderate PVR (grade B), and severe PVR (grade C or D) were aspirated during pars plana vitrectomy before infusion. In the current study, 129, 97 and 137 proteins were identified in vitreous of normal control, moderate and severe PVR, respectively. In PVR vitreous samples, complement components, serine proteinase inhibitors, and extracellular proteins were up-regulated or appeared, while normal cytoskeleton and metabolism proteins were down-regulated or disappeared. It was noteworthy that the proteins involved in transcription and translation regulation increased in vitreous with PVR. Among 102 PVR-specific proteins, kininogen 1 was specifically detected in both vitreous and the corresponding serum. Therefore, it can be concluded that PVR is a complicated pathology process with great amount of proteins involved in metabolism dysfunction, immune reactions, and cytoskeleton remolding. Kininogen 1 may be a candidate biomarker of PVR. Further investigations of these special proteins will provide additional targets for treatment or prevention of ocular proliferative diseases.

Comparative proteomic analysis of extracellular proteins reveals secretion of T-kininogen from vascular smooth muscle cells in response to incubation with s-enantiomer of propranolol.

Propranolol, a nonselective beta blocker, exerts blocking activity both on beta1 adrenoceptors and beta2 ones, with the S-enantiomer being more active than the R-enantiomer. The aim of the study was to investigate the secreted proteins with differential protein expression levels in culture medium of vascular smooth muscle cells (A7r5) incubated separately with individual enantiomers of propranolol using isobaric tags for relative and absolute quantitation (iTRAQ)-coupled two-dimensional LC-MS/MS approach. Our results indicated that secretion of T-kininogen by S-enantiomer of propranolol incubated cells was greatly enhanced as compared with that of R-enantiomer incubated cells or control cells. It can be inferred that the S-enantiomer of propranolol will induce more Ile-Ser-bradykinin (BK) (T-kinin), the vasoactive peptides. This therefore provides molecular evidence and possible link of T-kininogen with treatment of cardiovascular disease associated with propranolol treatment.

Gender differences in endothelial function and inflammatory markers along the occurrence of pathological events in stroke-prone rats.

Spontaneously hypertensive stroke-prone rats (SHRSP) feature an established model for human cerebrovascular disease. SHRSP, kept on a high-salt permissive diet (JPD), develop hypertension, renal and brain damage. In this report we compared the behavior of female and male SHRSP regarding the main aspects of their pathological condition. Brain abnormalities, detected by magnetic resonance imaging, developed spontaneously in males after 42+/-3 days, in females after 114+/-14 days from the start of JPD. Survival was >3-fold longer for females than for males. The development of brain damage was preceded, in both genders, by an inflammatory condition characterized by the accumulation in serum and urine of acute-phase proteins. The increase in thiostatin level was significantly lower and delayed in female in comparison to male SHRSP. During JPD female and male SHRSP developed massive proteinuria, its worsening being significantly slower in females. The alterations of vasculature-bound barriers in kidney and brain were connected with endothelial dysfunction and relative deficiency in nitric oxide (NO). In thoracic aortic rings, basal release of NO was significantly higher in female than in male SHRSP, both if receiving and if not receiving JPD. The gender differences in SHRSP thus appear to be connected to a more efficient control in females of inflammation and of endothelial dysfunction.

Tissue kallikrein and kininogen levels in fetoplacental tissues from normotensive pregnant women and women with pregnancy-induced hypertension.

OBJECTIVES: An imbalance of vasoconstrictor and vasodilator substances in the placenta has been postulated in the pathogenesis of pregnancy-induced hypertension (PIH). There is however little information available on the kallikrein-kinin system (KKS) in women with PIH. The aim of this study therefore was to determine tissue kallikrein and kininogen levels and their distribution patterns in fetoplacental tissues from both normotensive pregnant (NTP) women and women with PIH. STUDY DESIGN: The study group consisted of 24 women, 12 of whom had normal pregnancies, while 12 had PIH. Portions of amnion, chorion laeve, placental plate chorion, fetal placenta and maternal placenta were dissected from each freshly delivered placenta. Tissue kallikrein (total, active and inactive) and kininogen levels were estimated using a synthetic chromogenic substrate, S-2266 and an enzyme immunoassay, respectively. The data were analysed using Mann-Whitney U and Kruskal-Wallis tests. RESULTS: No significant differences were found for total, active and inactive tissue kallikrein levels in all fetoplacental tissues between both groups. However, kininogen levels were found to be significantly lower in chorion laeve, placental plate chorion, fetal placenta and maternal placenta from women with PIH when compared to those in similar tissues from NTP women. CONCLUSION: These findings suggest the presence of an abnormality in the kallikrein-kinin system in the placentas of women with PIH, which requires further study.

Pharmacological enhancement of the kallikrein-kinin system promotes anti-fibrotic responses in human mesangial cells.

The aim of the present study was to investigate whether pharmacological enhancement of the renal kallikrein-kinin system using the vasopeptidase inhibitor omapatrilat plays a direct role in modulating the fibrotic responses of human mesangial cells to injury. Treatment with 40 micromol/L omapatrilat was able to reduce macrophage-conditioned medium (MPCM)-induced fibronectin levels without affecting mRNA expression. MPCM injury also suppressed kallikrein and low molecular weight kininogen mRNA. Omapatrilat was able to attenuate this suppression. Bradykinin levels in contrast were increased by MPCM and treatment with omapatrilat further augmented levels. Co-incubation with the bradykinin B2 receptor antagonist HOE 140 attenuated the omapatrilat-induced lowering of fibronectin. Moreover, inhibition of cGMP release had a similar effect. Paradoxically, RT-PCR and Southern blotting demonstrated that bradykinin B2 receptor mRNA levels were down regulated in response to omapatrilat. Western blotting supported this data. Supernatant levels of tissue plasminogen activator (tPA), a product of bradykinin stimulation, were decreased by omapatrilat while cell associated tPA levels were increased. Matrix metalloproteinase-9 (MMP-9) mRNA expression was up regulated by omapatrilat treatment, although no difference in active zymogen levels was observed. In conclusion enhancement of kallikrein-kinin system appears to play a direct role in promoting anti-fibrotic responses in MPCM-injured human mesangial cells.

Acute phase protein induction by experimental inflammation in the salivary gland.

BACKGROUND: The submandibular gland (SMG) is a major salivary gland, which plays an important role in maintenance the oral health. In this study, we intended to explore the role of the SMG's defense system of the animals in which experimental inflammation is induced. METHODS: The levels of mRNAs for inflammation cytokines and acute phase proteins were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: The mRNAs for acute phase proteins were found to be increased in the SMG and extraorbital and intraorbital lacrimal gland (ELG and ILG) of rats at 24 h after subcutaneous injection of turpentine oil. The induction of mRNA for these inflammatory proteins by turpentine oil was preceded by a transient increase in the level of mRNAs for IL-1beta, IL-6 and TNF-alpha at 6 h after subcutaneous injection of the oil. Such cytokine induction was similarly seen by lipopolysaccharide (LPS) injection, and involvement of Toll-like receptor 4 (TLR4) was strongly suggested from experiment using C3H/HeJ mice, a TLR4-deficient mutant strain. CONCLUSION: The up-regulation of acute phase proteins and inflammation cytokines in the SMG, ELG and ILG by experimental inflammation suggests the existence of a strict defense system via the innate immune system in the SMG and other exocrine gland.

Tissue kallikrein is synthesized and secreted by human vascular endothelial cells.

The generation of kinins on the surface of vascular endothelium has been postulated in two pathways involving plasma kallikrein and tissue kallikrein; the former pathway has been well documented, but the latter is controversial. To clarify the presence of a kinin-generating system on endothelium, we examined whether human umbilical vein endothelial cells (HUVEC) synthesize and release tissue kallikrein in vitro. Kallikrein-like activity hydrolyzing a peptide Pro-Phe-Arg-4-methyl-coumaryl-7-amide was detected in the culture medium of HUVEC and was inhibited by aprotinin but not by soybean trypsin inhibitor. Western blotting of HUVEC medium using anti-human tissue kallikrein antibodies demonstrated the release of tissue kallikrein from HUVEC, and the reverse transcription-polymerase chain reaction (RT-PCR) followed by Southern blotting revealed the expression of tissue kallikrein mRNA in HUVEC. HUVEC metabolically labeled with [35S]methionine released radioactive proteins corresponding to tissue kallikrein. RT-PCR also showed the expression of low-molecular-weight kininogen (L-kininogen) mRNA in HUVEC. The cGMP levels in HUVEC were significantly elevated by the incubation with angiotensin converting enzyme inhibitor, lisinopril, and the elevation was completely inhibited by aprotinin or bradykinin B2-receptor antagonist, FR172357. These results suggest that the endothelial cells continuously release an active form of tissue kallikrein which enables generation of kinins on the vascular endothelium.

Bradykinin in the ascitic fluid of patients with liver cirrhosis.

Bradykinin, a nonapeptide with vasodilatory and permeabilizing activity, is generated through the cleavage of high-M(r) ('high-molecular-weight') kininogen by kallikrein, and its generation is facilitated by plasmin. In the ascitic fluid of patients with cirrhosis, there is massive cleavage of high-M(r) kininogen and activation of fibrinolysis, but bradykinin has never been measured directly. In the ascitic fluid of 24 patients with cirrhosis, we measured bradykinin-(1-9)-nonapeptide levels by RIA after liquid-phase and subsequent HPLC extraction, and those of its catabolic product bradykininin-(1-5)-pentapeptide by ELISA after liquid-phase extraction. Cleaved high-M(r) kininogen, activated factor XII and plasmin-antiplasmin complexes were measured in ascitic fluid and plasma. Plasma renin activity (PRA) was also determined. As a control, we also analysed plasma from 24 healthy subjects matched for sex and age with the patients. In the ascitic fluid from patients with cirrhosis, the median bradykinin-(1-9) concentration was 3.3 fmol/ml (range 0.2-29.0 fmol/ml), and the median bradykinin-(1-5) concentration was 210 fmol/ml (range 58-7825 fmol/ml). The levels of bradykinin-(1-5) in ascitic fluid were higher in patients with refractory ascites [median 1091 fmol/ml (range 58-7825 fmol/ml)] than in patients with responsive ascites [134 fmol/ml (72-1084 fmol/ml)] (P=0.010). Ascitic fluid levels of bradykinin-(1-9) were not related to the severity of ascites. PRA was higher in patients with refractory ascites [23.0 ng x h(-1) x ml(-1) (7.9-80.0 ng.h(-1).ml(-1))] than in patients with responsive ascites [6.9 ng x h(-1) x ml(-1) (0.9-29.4 ng x h(-1) x ml(-1))] (P=0.002). In ascitic fluid, 48% (19-68%) of high-M(r) kininogen was cleaved, and plasmin-antiplasmin complexes were more concentrated than in plasma (P=0.0001). In conclusion, in the ascitic fluid of patients with cirrhosis, both bradykinin-(1-9) and bradykinin-(1-5) are present, with cleavage of high-M(r) kininogen and activation of fibrinolysis. The highest levels of the long-lived metabolite bradykinin-(1-5) were found in the ascitic fluid of patients with refractory ascites and high PRA. Activation of the kinin system may therefore be involved in decompensating cirrhosis, but a cause-effect relationship remains to be established.

[The assessment of expression of kininogen and bradykinin receptors genes in different vulvar pathologies by QPCR (TaqMan)]. / Ocena ekspresji genów kodujacych kininogen oraz receptory kinin w róznych stanach patologii sromu metoda QPCR (TaqMan).

OBJECTIVES: In different vulvar pathologies inflammatory process and pain are often observed. In these processes, kinins, released from kininogen, play an important role. Their effects are mediated by at least two types of bradykinin receptors--B1 and B2. B1 receptor appears in certain pathological states, B2 is widely distributed in normal tissues. The expression of genes coding kininogen, B1 and B2 receptors can be a very sensitive marker of tissue pathology. DESIGN: In the present study, the analysis of expression of genes coding kininogen, B1 and B2 was performed. The relation between the analysed genes expression and the pathology stage was analysed. MATERIALS AND METHODS: The specimens from condylomata accuminata, vulvar cancer and surgical margin were analysed. The number of DNA and mRNA copies of beta-actine, kininogen, B1 and B2 were examined basing on Q-PCR standard curves for beta-actine by use of Perkin Elmer-kit and the sequence detector ABI PRISM 7700-Taq Man application. RESULTS: In condylomata accuminata the high expression of mRNA of kininogen, B1 and B2 was found, while in vulvar cancer tissue, the expression of analysed genes was low. In the tissue from the tumour center, the lowest kinin genes expression was stated. CONCLUSIONS: The absence of kininogen and B2 mRNA expression characterised vulvar cancer tissue. The profile of expression of kininogen and its receptor genes can be a useful marker in the assessment of vulvar cancer surgical margin.

Different mechanisms define the antiadhesive function of high molecular weight kininogen in integrin- and urokinase receptor-dependent interactions.

Proteolytic cleavage of single-chain high molecular weight kininogen (HK) by kallikrein releases the short-lived vasodilator bradykinin and leaves behind 2-chain high molecular weight kininogen (HKa) that has been previously reported to exert antiadhesive properties as well as to bind to the urokinase receptor (uPAR) on endothelial cells. In this study we defined the molecular mechanisms for the antiadhesive effects of HKa related to disruption of integrin- and uPAR-mediated cellular interactions. Vitronectin (VN) but not fibrinogen or fibronectin-dependent alphavbeta(3) integrin-mediated adhesion of endothelial cells was blocked by HKa or its isolated domain 5. In a purified system, HKa but not HK competed for the interaction of VN with alphavbeta(3) integrin, because HKa and the isolated domain 5 but not HK bound to both multimeric and native VN in a Zn(2+)-dependent manner. The interaction between HKa or domain 5 with VN was prevented by heparin, plasminogen activator inhibitor-1, and a recombinant glutathione-S-transferase (GST)-fusion peptide GST-VN (1-77) consisting of the amino terminal portion of VN (amino acids 1-77), but not by a cyclic arginyl-glycyl-aspartyl peptide, indicating that HKa interacts with the amino terminal portion of VN ("somatomedin B region"). Furthermore, we have confirmed that HKa but not HK bound to uPAR and to the truncated 2-domain form of uPAR lacking domain 1 in a Zn(2+)-dependent manner. Through these interactions, HKa or its recombinant His-Gly-Lys-rich domain 5 completely inhibited the uPAR-dependent adhesion of myelomonocytic U937 cells and uPAR-transfected BAF-3 cells to VN and thereby promoted cell detachment. By immunogold electron microscopy, both VN and HK/HKa were found to be colocalized in sections from human atherosclerotic coronary artery, indicating that the described interactions are likely to take place in vivo. Taken together, HK and HKa inhibit different VN-responsive adhesion receptor systems and may thereby influence endothelial cell- or leukocyte-related interactions in the vasculature, particularly under inflammatory conditions. (Blood. 2000;96:514-522)

Altered cardiac tissue and plasma kininogen levels in hypertensive and diabetic rats.

The present investigation was aimed at evaluating the cardiac and total plasma kininogen levels, as well as LVWT in hypertensive and diabetic rats. STZ-induced diabetes produced a significant (P < 0.001) rise in mean arterial blood pressure (BP). The LVWT increased (P < 0.001) in SHR with and without diabetes) and diabetic WKYR. The cardiac tissue, as well as total plasma kininogen levels fell significantly (P < 0.001) in diabetic WKYR and SHR with and without diabetes compared to the control WKYR. These findings suggest that reduced kininogen levels may indicate a deficiency in kinin generation in the heart and in the peripheral circulation in diabetic and hypertensive rats. This effect may contribute to the development of LVH.

Immunohistochemical localization of kininogen in rat spinal cord and brain.

Kininogen localization has been determined by immunocytochemistry in rat spinal cord and brain using a kinin-directed kininogen monoclonal antibody. In the spinal cord, there were immunostained neurons and fibers in laminae I, II, VII, and IX, intensely stained fibers in the superficial layers of the dorsal horn, and immunoreactive glial and endothelial cells. Small neurons, satellite cells, and Schwann cells immunostained distinctly in the dorsal root ganglion. In the brain stem, there were immunoreactive neurons and fibers in the tractus solitarius and nucleus, trigeminal spinal tract and nuclei, periaqueductal gray matter, vestibular nuclei, cochlear nuclei, trapezoid body, medial geniculate nucleus, and red nucleus. Immunostained neurons and fibers were also found in cerebellum (dentate nucleus), cerebral cortex (layers III and V), hippocampus (pyramidal cell layer), and corpus callosum. Glia and endothelial cells stained in all brain regions. The widespread location of kininogen in neurons and their processes, as well as in glial and endothelial cells, indicates more than one functional role, including those proposed as a mediator, a calpain inhibitor, and a kinin precursor, in a variety of neural activities and responses.

Lack of clinically significant contact system activation during platelet concentrate filtration by leukocyte removal filters.

When blood (plasma) contacts certain foreign surfaces, factor XII can activate and trigger a series of reactions leading to cleavage of kininogens with subsequent release of bradykinin. In this study, we investigated two different widely used leukocyte removal filters, Pall PXL8K (A) and Asahi PLS-5A (B), to test whether clinically significant contact activation occurred during leukodepletion of platelet-rich plasma (PRP). Kininogens were measured by particle concentration fluorescence immunoassay (PCFIA), which can detect cleavage of high and low molecular weight kininogens (HK and LK), the parent molecules of bradykinin, to determine if contact activation had occurred. A slight, nonsignificant decrease in HK and LK was observed with filter A after the first 5 mL was filtered that returned to prefiltration levels by the end of the filtration. Specific TotK (the combined measurement of HK and LK heavy chains divided by plasma protein concentration) showed a small, significant decrease with filter A after the first 5 mL of platelet concentrates was filtered that returned to prefiltration levels by the end of the filtration. There were no significant increases or decreases in the cleaved kininogen index (CKI), an index of HK proteolytic activation or HK and LK destruction (with release of bradykinin). These data suggest that small amounts of both HK and LK initially adsorb to filter A and then desorb, primarily intact. These data also indicate that no significant contact activation, as measured by PCFIA, occurs during leukodepletion of platelet concentrates with either filter A or B.